WO2021163642A2 - Lymphocytes t modifiés par un gène foxp3 à base de crispr et précurseurs de cellules souches hématopoïétiques permettant de traiter des patients atteints de syndrome ipex - Google Patents

Lymphocytes t modifiés par un gène foxp3 à base de crispr et précurseurs de cellules souches hématopoïétiques permettant de traiter des patients atteints de syndrome ipex Download PDF

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WO2021163642A2
WO2021163642A2 PCT/US2021/018057 US2021018057W WO2021163642A2 WO 2021163642 A2 WO2021163642 A2 WO 2021163642A2 US 2021018057 W US2021018057 W US 2021018057W WO 2021163642 A2 WO2021163642 A2 WO 2021163642A2
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foxp3
cells
edited
homology
gene
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WO2021163642A3 (fr
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Rosa Bacchetta
Maria-Grazia Roncarolo
Matthew Porteus
Marianne GOODWIN
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The Board Of Trustees Of The Leland Stanford Junior University
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Priority to EP21753306.6A priority Critical patent/EP4103591A2/fr
Priority to CA3168089A priority patent/CA3168089A1/fr
Priority to US17/760,264 priority patent/US20230081343A1/en
Publication of WO2021163642A2 publication Critical patent/WO2021163642A2/fr
Publication of WO2021163642A3 publication Critical patent/WO2021163642A3/fr

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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C12N5/0637—Immunosuppressive T lymphocytes, e.g. regulatory T cells or Treg
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4621—Cellular immunotherapy characterized by the effect or the function of the cells immunosuppressive or immunotolerising
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/46433—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
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    • C12N2510/00—Genetically modified cells

Definitions

  • Primary immunodeficiencies comprise a group of genetic immune diseases, which typically present with recurrent infections, but may instead manifest with predominant autoimmunity. Over 350 monogenic immune diseases have been described to date, and this number has been rapidly increasing with technological advances in DNA sequencing and expanding accessibility of genetic screening.
  • the prototype of genetic autoimmunity is immune dysregulation, polyendocrinopathy, enteropathy, immune dysregulation polyendocrinopathy enteropathy X- linked (IPEX) syndrome, which is a severe X-linked disease with early onset.
  • IPEX immune dysregulation polyendocrinopathy enteropathy X- linked
  • the most frequent autoimmune manifestations of IPEX syndrome include type 1 diabetes, eczema, and life-threatening enteropathy. Other common autoimmune manifestations include cytopenia, autoimmune hepatitis, and thyroiditis.
  • IPEX syndrome is classified as a Tregopathy, a class of diseases that selectively affect the function of regulatory T cells (Tregs), and in the case of IPEX syndrome, CD4 + CD25 h ' 9h FOXP3 + Tregs.
  • Tregs regulatory T cells
  • CD4 + CD25 h ' 9h FOXP3 + Tregs CD4 + CD25 h ' 9h FOXP3 + Tregs.
  • non-functional Tregs are produced that are unable to prevent the development of autoimmunity or allergy because they lack the ability to suppress the function and proliferation of effector T (Teff) cells.
  • Teff effector T
  • IPEX patients are treated with pharmacological immunosuppression, which has only partial efficacy in the acute phase of the disease and cannot prevent long-term disease progression. Furthermore, administration of immunosuppressive drugs carries the risk of severe side effects associated with toxicity and susceptibility to infections.
  • the only curative treatment available for IPEX is allogeneic hematopoietic stem cell transplantation. However, many patients do not find a suitable donor or suffer from transplant related complications.
  • IPEX syndrome is caused by mutations in the forkhead box protein 3 ( FOXP3 ) gene, and over 70 unique mutations throughout the FOXP3 locus have been identified.
  • FOXP3 is a master transcription factor required for the function of Tregs, which upregulates Treg- associated markers, such as CD25 and CTLA4, and represses proinflammatory cytokine production. While Tregs rely on constitutive FOXP3 expression, Teff cells transiently express FOXP3 following TCR activation. This cell-type specific regulation is a result of a complex network of promoter and enhancer elements.
  • Treg-specific demethylated region TSDR
  • Teff cells require transient FOXP3 expression for intrinsic regulation of proliferation, cytokine production, and TCR signaling.
  • IPEX impairment of both Teff and Treg function underlies IPEX syndrome pathology.
  • FOXP3 pre-mRNA is alternatively spliced, and the two predominate spliced isoforms are the full length (FOXP3F L ) isoform and a shorter version that lacks exon 2 (FOXP3 ⁇ E2 ).
  • the FOXPCF L and FOXP ⁇ 2 isoforms each represents roughly half of the FOXP3 expressed, however the proportion is skewed in different cell activation states and in a number of inflammatory diseases.
  • Causative IPEX mutations in exon 2 have been described, with a subset of patients presenting with milder clinical phenotypes. Because these mutations spare the FOXP ⁇ 2 isoform, it has been suggested that FOXPSf ⁇ 2 can partially compensate for FO P3 FL loss, but that FO P3 FL is required for complete Treg and Teff cell function and prevention of IPEX syndrome.
  • IPEX syndrome is a monogenic immune disease caused by mutations in FOXP3
  • gene therapy could be a useful approach to treat the disease.
  • a FOXP3 gene delivery protocol for ex vivo generation of genetically engineered Tregs has been developed that uses lentiviral vector (LV)-mediated delivery of copy of the complementary DNA (cDNA) of the full length isoform of FOXP3. Because this vector expresses FOXP3 under a constitutive promoter, EF1a, it is able to convert IPEX patient conventional CD4+ T cells into potent Treg-like suppressor cells.
  • LV lentiviral vector
  • cDNA complementary DNA
  • IPEX syndrome clinical manifestations recapitulate those occurring in many other autoimmune diseases more commonly observed in the general population. These autoimmune diseases are not due to monogenic defects but rather results from genetic predisposition and environmental co-morbidity factors. Nevertheless, abnormal function or number of Treg cells are involved in their pathogenesis and Treg cell immunotherapy is envisaged as innovative treatment for these diseases.
  • IPEX is a model of Primary Immune Regulatory Disorders (PIRD) in which Treg and Teff can be dysfunctional because of mutations in genes relevant for both and characterized by immune dysregulation.
  • PIRD Primary Immune Regulatory Disorders
  • the method provided herein for the correction of FOXP3 can be applied to many other PIRD. The present disclosure addresses this condition.
  • a method for restoring functional regulatory T cell activity to an individual in need thereof e.g. an individual suffering from immune dysregulation polyendocrinopathy enteropathy X-linked (IPEX) syndrome.
  • the gene edited hematopoietic cells comprise a site-directed gene correction of the FOXP3 gene.
  • the cells are autologous to a recipient, for example where the cells are isolated from a patient sample, gene corrected ex vivo, and reintroduced to the recipient.
  • the cells are allogeneic to the recipient.
  • the recipient suffers from IPEX.
  • the hematopoietic cells are hematopoietic stem or progenitor cells (HSPC), e.g. CD34 + human hematopoietic stem cells, lymphoid progenitor cells, etc., which may be isolated from peripheral blood, cord blood, bone marrow, etc. as known in the art.
  • the hematopoietic cells are T cells, which may be isolated from patient samples, for example by selection for positive expression of CD4, CD25, etc. or may be differentiated from HSPC.
  • the T cells are CD4 + T effector (Teff) cells.
  • the T cells are CD4 + regulatory T cells (Treg).
  • the cells produced by this method may be referred to herein as hematopoietic edFOXP3 cells, e.g. HSPC edF0XP3 CD4 edFOXP3 T cells, etc.
  • FOXP3 engineered human hematopoietic cells are produced by gene editing of hematopoietic cells ex vivo by CRISPR/Cas effector gene editing.
  • the gene editing method may comprise introducing into the targeted cell the components: sgRNA complexed to a Cas protein as an RNP system; and a FOXP3 homology donor vector.
  • the FOXP3 homology donor vector comprises a coding sequence for FOXP3, usually a full-length coding sequence.
  • the coding sequence may be a cDNA, or may comprise one or more introns.
  • the coding sequence can be modified, or diverged, to incorporate synonymous mutations at the nucleotide level according to the redundant codon usage system, to prevent premature recombination while still encoding for a wild-type protein.
  • the FOXP3 sequence encodes a functional, wild-type FOXP3 protein, although for research purposes a mutated form may be encoded.
  • the FOXP3 protein may be one of the FOXP3 isoforms FO P3 FL (SEQ ID NO:1 ) or FOXP3P E2 (SEQ ID NO:2).
  • the FOXP3 coding sequence is generally not linked to a promoter in the vector, and is expressed in the cell by the native FOXP3 promoter. This is a fundamental advantage of this approach, that in the case of the FOXP3 gene editing allows endogenously regulated expression which occur differentially in Treg and Teff.
  • the FOXP3 coding sequence may be operably linked to a polyadenylation sequence, including without limitation BGH polyadenylation signal.
  • the homology vector optionally comprises a marker sequence, including without limitation a truncated nerve growth factor receptor (tNGFR) operably linked to a promoter, e.g. the phosphoglycerate kinase 1 (PGK) promoter.
  • tNGFR truncated nerve growth factor receptor
  • PGK phosphoglycerate kinase 1
  • the homology donor vector further comprises a 5’ and a 3’ arm with homology to the FOXP3 locus (chromosomal site); where the homology arms may be centered on the cut site of the sgRNA.
  • the recombinant FOXP3 homology donor vector comprises the nucleotide sequence of SEQ ID NO:3 or SEQ ID NO:4, or a sequence having at least about 80- 100% sequence identity thereto, for example at about 95% sequence identity, including any percent identity within this range, such as 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, wherein the recombinant FOXP3 homology donor vector is capable of gene correcting a mutated FOXP3 sequence in a hematopoietic cell of interest.
  • the sgRNA comprises 2'-0-methyl 3'phosphorothioate (MS) chemical modifications at the terminal nucleotides.
  • the Cas protein is Cas9 protein.
  • the sgRNA comprises the sequence set forth in Table 1 , e.g. SEQ ID NO:5-12, and in some embodiments comprises SEQ ID NO:6.
  • a method of producing CD4 edFOXP3 T cells comprising: a) obtaining a biological cell sample comprising one of HSPC, lymphoid progenitor cells, or CD4 + T lymphocytes from a subject; b) gene editing the cells with CRISPR/Cas9 and FOXP3 homology donor vectors described herein; and c) culturing the cells under conditions suitable for expression of the FOXP3, wherein CD4 + T lymphocytes are edited into CD4 edFOXP3 T cells.
  • the cells targeted for gene editing are HSPC or lymphoid progenitor cells
  • the cells may be differentiated into CD4 + T cells.
  • HSPC are transplanted to a recipient and differentiated into Treg and Teff CD4 edFOXP3 T cells in vivo.
  • the biological cell sample can be any sample comprising targeted hematopoietic cells, e.g. peripheral blood, bone marrow, etc. Isolation of HSPC may utilize mobilized peripheral blood, as known in the art (see, for example, Karpova et al. (1019) F1000Res.; 8: F1000 Faculty Rev-2125).
  • the method further comprises isolating the targeted cells, e.g. HSPC, lymphoid progenitor cells, CD4 + T lymphocytes, etc. from the biological sample.
  • the method further comprises substantially purifying the cells after gene editing.
  • the gene edited cells are substantially purified by positive selection for a cell surface marker encoded by the homology donor vector.
  • the cell surface marker is a truncated nerve growth factor receptor (tNGFR)
  • the gene edited cells can be substantially purified by positive selection for the tNGFR cell surface marker using for example and without limitation, immunomagnetic separation or flow cytometry.
  • the method further comprises culturing the hematopoietic cells during and after the gene editing process.
  • the method further comprises culturing CD4 edFOXP3 T cells.
  • the method further comprises adding IL-2 to a culture of CD4 edFOXP3 T cells to expand the number of CD4 edFOXP3 T cells in the culture.
  • gene edited hematopoietic cells produced by the methods described herein are provided, for example a population of isolated HSPC edF0XP3 ; CD4 edFOXP3 T cells; etc.
  • a composition comprising FOXP3 gene edited cells produced by the methods described herein are provided for use in treatment of an inflammatory condition.
  • a composition of the FOXP3 gene edited cells is substantially purified free of other cells.
  • the composition further comprises a pharmaceutically acceptable excipient.
  • a composition comprising the FOXP3 gene edited cells for use in treatment of IPEX syndrome is provided.
  • a method of treating an inflammatory condition in a subject comprising administering a therapeutically effective amount of a composition comprising FOXP3 gene edited cells produced by the methods described herein to the subject.
  • the composition is generally administered in an amount sufficient to reduce inflammation in the subject.
  • a method of adoptive cellular immunotherapy for treating an inflammatory condition comprising: a) obtaining a biological cell sample comprising one of HSPC, lymphoid progenitor cells, or CD4 + T lymphocytes from a subject; b) gene editing the cells with CRISPR/Cas9 and FOXP3 homology donor vectors described herein; c) administering a therapeutically effective amount of the cells to the subject.
  • the cells targeted for gene editing are HSPC or lymphoid progenitor cells
  • the cells may be differentiated into CD4 edFOXP3 T cells.
  • HSPC are transplanted to a recipient and differentiated into CD4 edFOXP3 T cells in vivo.
  • the biological cell sample can be any sample comprising targeted hematopoietic cells, e.g. peripheral blood, cord blood, bone marrow, etc.
  • the method further comprises isolating the targeted cells, e.g. HSPC, lymphoid progenitor cells, CD4 + T lymphocytes, etc. from the biological sample.
  • the methods described herein can be used to treat inflammatory conditions, including for example, without limitation, Treg deficiency, autoimmune disorders, allergies, graft-versus-host disease, and transplant rejection.
  • the Treg deficiency/autoimmune disorder is IPEX syndrome.
  • a method of treating immune dysregulation polyendocrinopathy enteropathy X-linked (IPEX) syndrome comprising administering a therapeutically effective amount of a composition comprising autologous HSPC edF0XP3 or CD4 edF ox P3 T cells to the subject, as described herein.
  • FOXP3 gene edited cells produced by the methods described herein may be administered by any suitable mode of administration.
  • the cells are administered intravenously or intra-arterially.
  • the cells are administered locally at a site of inflammation.
  • the cells are administered locally at a site of a tissue or organ transplant.
  • Method of transplantation for hematopoietic stem cells may use myeloablative or non-myeloablative conditioning, including antibody-mediated conditioning, e.g. as disclosed in US Patent nos.10,882,915; 10,111 ,966; 10,406,179; US Patent publications 20200369767; 20200129557; and 20180214524, each herein specifically incorporated by reference.
  • FIG. 1 The FOXP3 locus is precisely targeted using the CRISPR system in primary HSPCs and T cells.
  • A Schematic representation of CRISPR-based editing of the FOXP3 gene showing the CRISPR cut site in first coding exon, E1 (exons depicted by grey boxes separated by lines representing introns; non-coding exon E-1 , enhancer with TSDR).
  • E1 exons depicted by grey boxes separated by lines representing introns; non-coding exon E-1 , enhancer with TSDR.
  • a zoomed- in view of the sgRNA binding site relative to the start codon, PAM site, and cleavage site is shown.
  • CRISPR efficiency measured by TIDE analysis to detect indel mutations created by NHEJ- mediated DNA repair CRISPR efficiency measured by TIDE analysis to detect indel mutations created by NHEJ- mediated DNA repair.
  • C Experimental method for editing of HSPCs and T cells with functional readouts listed.
  • D CRISPR cutting efficiency in CD34+ HSPCs and CD4+ Tcells quantified by TIDE analysis for detection of indel mutations created by the NHEJ repair pathway.
  • FIG. 1 CRISPR combined with a rAAV6 homology donor enables precise HDR- mediated FOXP3 cDNA transgene insertion into the endogenous locus.
  • A Editing observed at the DNA level by an in-out PCR strategy that uses a primer inside the inserted divergent cDNA construct and a second primer outside of the 5’ arm of homology. Control band represents unmodified region in the FOXP3 gene as a positive control for the presence of genomic DNA. PCR using in-out primers resulted in band only present in samples in which the cDNA was inserted (FL cDNA) and not in FOXP3 knockout (KO) or mock treated samples.
  • Negative control mock treated cells nucleofected with PBS in place of CRISPR and transduced with rAAV6 -FOXP3 donor. Edited cells enriched using tNGFR selection and purity shown by flow cytometry.
  • D Venn diagram showing overlap in predicted off-target (OT) sites identified by COSMID in silico prediction and GUIDE-seq DSB capture. All predicted sites tested by next generation sequencing (NGS) in edited CD34+ HSPCs derived from cord blood (CB), and validated by NGS in edited bone marrow (BM)- derived HSPCs.
  • NGS next generation sequencing
  • BM bone marrow
  • FIG. 3 FOXP3 edited Tregs express FOXP3 protein and display characteristic in vitro phenotype and function.
  • A Quantification of FOXP3 protein expression in the MT-2 Treg cell line by flow cytometry, showing median fluorescent intensity (MFI) values for one representative experiment out of three performed.
  • B FOXP3 homology donor constructs designed to improve FOXP3 protein expression, including further codon optimization (FOXP3 FLco ) and addition of a WPRE element (FOXP3 fl ⁇ w ).
  • C Flow cytometry for FOXP3 in MT-2 cells comparing the different donor constructs.
  • FIG. 4 FOXP3 editing of Teff cells preserves physiological regulation of FOXP3 expression and in vitro function.
  • A Flow cytometry time course showing kinetics of FOXP3 expression in non-activated Teff cells and activated Teff cells on subsequent days post- activation (d3, d6, d14), comparing WT unmodified, WT mock treated, and FO P3 FLcoW edited Teff cells.
  • B Cytokine production in WT and FOXP3 gene edited Teff cells determined by ELISA.
  • (C) Teff cell proliferation in response to activation measured by the proliferation assay. Shown are flow cytometry plots of CFSE dye stained Teff cells with progressive dilution of dye as proliferation progresses from non-activated to d2 and d3 post-activation with anti-CD3/28 dynabeads. Comparison of proliferation rates in response to activation with a bead:cell ratio of 1 :100 and 1 :25. Quantification of average proliferative response of Teff cells from proliferation assay at day 3 shown to the right, comparing different doses of activation beads (mean ⁇ SD, n 3).
  • FIG. 5 CRISPR-based editing enables FOXP3 gene correction in IPEX patient cells.
  • A Schematic of FOXP3 gene highlighting mutations of patients involved in this study.
  • B Editing of IPEX and HD T cells observed at the DNA level by in-out PCR strategy. Forward primer is in the tNGFR cassette and the reverse primer is in the FOXP3 gene locus outside the 3’ arm of homology. Positive and negative fractions after tNGFR enrichment (+/-) analyzed by PCR.
  • C Flow cytometry plots of tNGFR staining.
  • FIG. 6 FOXP3 edited HSPCs undergo multilineage hematopoietic differentiation and engraft in vivo.
  • A Differentiation potential of edited HSPCs tested by the in vitro colony forming unit (CFU) assay.
  • CFU-E mature erythroid progenitors
  • CFU-GEMM granulocyte, erythrocyte, macrophage, megakaryocyte
  • BFU-E primary erythroid progenitors
  • CFU-GM granulocyte and macrophage progenitors
  • FOXP3 edited samples were divided into tNGFR+ and tNGFR- gates for comparability.
  • E Quantification of human hematopoietic lineages by flow cytometry with each symbol representing a single mouse (mean ⁇ SD).
  • the CD8+, CD4+, and CD4+CD8+ double positive (DP) populations were gated out of CD3+ T cells.
  • the CD25+FOXP3+, naive CD45RA+, and memory CD45RA- populations were gated out of CD4+ single positive T cell subset.
  • FIG. 7 The CRISPR system allows for precise FOXP3 gene modification.
  • (Corresponding to Figure 1) (A) Schematic representation of the edited FOXP3 allele after HDR- mediated insertion of a cDNA encoding the alternatively spliced isoform of FOXP3 lacking exon2 (dE2, FOXP3P E2 , top construct). Construct includes the inserted tNGFR marker gene under the constitutive promoter, PGK, allowing marking of all edited cells.
  • the FOXP3 knockout allele (KO, FOXP 0 , bottom construct) created by insertion of the tNGFR marker gene without a FOXP3 cDNA.
  • the tNGFR marker cassette flanked by polyadenylation signals (pA) to terminate mRNA processing and block expression of the downstream FOXP3 gene elements, creating FOXP3 knockout while marking edited cells.
  • pA polyadenylation signals
  • B The sequence of CRISPR sgRNA binding sites in exon 1 of the FOXP3 gene relative to the start codon (red). The cut site of each sgRNA is underlined. The sgRNAs were tested either individually (sg1 , sg2, sg3, and sg4) or as pairs (sg5&6 and sg7&8).
  • FIG. 8 The FOXP3 gene is precisely edited using CRISPR-mediated homology directed repair.
  • (Corresponding to Figure 2) (A) Precise targeting of the FOXP3 gene shown by an alternative in-out PCR strategy with forward primer (FP) in tNGFR and the reverse primer (RP) in the endogenous FOXP3 gene outside of the 3’ arm of homology. Band representing successful recombination observed from FO P3 FL and FOXP ⁇ 0 gene edited HSPCs, both of which contain the tNFGR cassettes (adjacent lanes represent biological replicates). Control band targeting non-modified FOXP3 region.
  • FP forward primer
  • RP reverse primer
  • FIG. 9 Tregs and Teff cell populations are effectively separated prior to CRISPR- based editing.
  • A Purity of the fractionated Treg and Teff cell samples from peripheral blood shown by flow cytometry after anti-CD25 magnetic bead separation using two serial columns (CD25++). Representative flow cytometry plots showing the total population of CD4+ T cells prior to separation (left panel); Teff cell CD25- fraction (middle panel); and CD25++ Treg-enriched fraction (right panel) stained for Tregs in two parallel gating strategies.
  • B Frequency of TSDR demethylated Tregs by epigenetic bisulfite qPCR. Shown are CD25- fraction after anti-CD25 magnetic bead separation enriched for Teff cells, CD25++ fraction enriched for Tregs, and MT-2 Treg cell line for comparison.
  • FIG. 10 The FOXP3 gene is knocked-in and knocked out using CRISPR-based homologous recombination.
  • FIG. 3 A Example flow cytometry plot showing that MT-2 Tregs edited with the FL construct co-express FOXP3 and tNGFR. Overlay contains negative control sample that is 98% double negative for FOXP3 and tNGFR (FOXP3- tNGFR-), WT mock treated cells that are 98% FOXP3+ tNGFR-, and FL cDNA edited cells that are 93% double positive (FOXP3+tNGFR+).
  • FIG. 11 Tregs edited with different constructs display comparable amounts of FOXP3 function and in vitro suppressive capacity.
  • MFI median fluorescent intensity
  • B Suppression assay comparing Tregs edited with cDNAs of the two FOXP3 isoforms, FOXP3F LcoW and FOXP ⁇ 2 . The percent calculated suppression shown to the left.
  • C Suppression assay testing the function of Tregs from two healthy donors (HD) edited with FOXPCF 1 and FOXP3 LcoVJ . The calculated percent suppression of CFSE-labeled stimulated responders (R * ) is shown to the left. As a negative control, cultured Teff cells from a parallel FOXP3 editing experiment were used in place of Tregs and were shown to not be suppressive (N/A).
  • D Suppression assay demonstrating that FQXF’3 FLcoW edited Teff cells lack suppressive function as anticipated. The proliferation rate of stimulated responders (R * ) is similar to that of responders co-cultured with WT mock treated or FOXP3F LcoVJ edited Teff cells.
  • FIG. 13 FOXP3 edited HSPCs retain multi-lineage engraftment and differentiation potential.
  • A Phenotypic analysis of edited and control HSPCs pre-injection by flow cytometry, evaluating editing rates (tNGFR+), CD34+ purity, and different HSPC subsets including lymphoid-primed multipotent progenitors (LMPP, CD34+CD38- CD45RA+CD90-/v), multipotent progenitors (MPP, CD34+CD38-CD45RA-CD90-), and HSCs (CD34+CD38-CD45RA-CD90+).
  • B Survival curve of mice engrafted with HSPCs from 3 experimental conditions over time.
  • C Persistence of edited tNGFR+ cells engrafted in the hu- mouse at wk 14 demonstrated by flow cytometry.
  • D Quantification of tNGFR+ rates in hu- mouse bone marrow at wk 14 showing different cord blood HSPC donors.
  • E Genomic analysis showing percentage of cells with unmodified wild-type FOXP3 alleles (blue), alleles edited by NHEJ and containing indel mutations (gray, TIDE analysis), and alleles edited by HDR (red, ddPCR in-out PCR).
  • the present disclosure provides genetically modified cells and methods of producing such cells. Also provided are methods of editing the genome of such cells.
  • the genetically modified cells of the disclosure are genetically modified such that their genome includes an integrated heterologous FOXP3 coding nucleic acid at one or more positions within the genome, operably linked to the native FOXP3 promoter present in the genome.
  • the newly integrated FOXP3 sequence replaces a mutated sequence present in the genome with a wild- type sequence.
  • a CRISPR/Cas protein (also referred to herein as a CRISPR/Cas endonuclease) interacts with (binds to) a corresponding guide RNA to form a ribonucleoprotein (RNP) complex (referred to herein as a CRISPR/Cas complex) that is targeted to a particular site (a target sequence) in a target genome via base pairing between the guide RNA and a target sequence within the target genome.
  • RNP ribonucleoprotein
  • a guide RNA includes (i) a nucleotide sequence (a guide sequence) that is complementary to a sequence (the target site) of a target DNA and (ii) a protein-binding region that includes a double stranded RNA (dsRNA) duplex and bind to a corresponding CRISPR/Cas protein.
  • the guide RNA can be readily modified in order to target any desired sequence within a target genome (by modifying the guide sequence). Suitable guide RNA sequences are provided, for example, in Table 1.
  • a wild type CRISPR/Cas protein (e.g., a Cas9 protein) normally has nuclease activity that cleaves a target nucleic acid (e.g., a double stranded DNA (dsDNA)) at a target site defined by the region of complementarity between the guide sequence of the guide RNA and the target nucleic acid.
  • a target nucleic acid e.g., a double stranded DNA (dsDNA)
  • dsDNA double stranded DNA
  • CRISPR/Cas protein includes wild type CRISPR/Cas proteins, and also variant CRISPR/Cas proteins, e.g., CRISPR/Cas proteins with one or more mutations in a catalytic domain rendering the protein a nickase.
  • a heterologous nucleic acid is integrated into the genome of a cell, which for the purposes of the present disclosure is typically a human hematopoietic cell.
  • the heterologous nucleic acid can be any desired length, but will comprise a FOXP3 coding sequence.
  • the term “heterologous” is a relative term. In some cases, the heterologous nucleic acid is heterologous to the genome because the exact sequence is present nowhere in the genome except for where the nucleic acid has integrated, although a highly similar sequence is usually present.
  • a nucleic acid that is integrated into the genome at one or more positions includes a CRISPR/Cas target sequence.
  • two or more nucleic acids are integrated into two or more different positions within the same locus (e.g., flanking a nucleotide sequence encoding a protein and/or an RNA, or a transcription control element). For example, both isoforms of FOXP3 may be integrated.
  • locus refers to a position (which position can be particular base pair location, or can be a range of from one base pair to another) within a genome of interest.
  • a locus can be a particular base pair position.
  • a locus can be a range of base pair positions, e.g., the position in the genome that codes a particular protein or RNA that is transcribed (as an illustrative example, the FOXP3 locus is a protein-coding locus that is transcribed and encodes the FOXP3 protein).
  • the term protein-coding locus or RNA-coding locus generally includes the transcriptional control sequences that influence transcription of the locus.
  • the term “protein-coding locus” not only refers to the nucleotide sequences that have an open reading frame (ORF) and directly encode the protein, but also the promoter, the 5’ UTR, the 3’ UTR, etc.
  • a target DNA e.g., genomic DNA
  • a CRISP/Cas protein e.g., Cas9
  • target site e.g., genomic DNA
  • CRISPR/Cas target site or “CRISPR/Cas target sequence” are used interchangeably herein to refer to a nucleic acid sequence present in a target DNA (e.g., genomic DNA of a cell) to which a CRISPR/Cas guide RNA can bind, allowing cleavage of the target DNA by the CRISPR/Cas endonuclease.
  • a target sequence can be any desired length and, in some cases, can depend upon the type of CRISPR/Cas guide RNA and CRISPR/Cas protein that will be used to target the target sequence.
  • a feature that renders the target sequence functional is that it is adjacent to a protospacer adjacent motif (PAM), also referred to as a “PAM sequence.”
  • PAM protospacer adjacent motif
  • the CRISPR/Cas target sequence is adjacent to a PAM.
  • the PAM can be present at that position in the genome prior to the integration (e.g., the nucleic acid can be integrated such that the CRISPR/Cas target sequence is inserted next to the PAM that was already present in the genome.
  • the PAM is not present at the desired position in the genome, and the PAM is instead present on the nucleic acid to be integrated.
  • a heterologous nucleic acid would therefore include the CRISPR/Cas target sequence adjacent to a PAM sequence, and both the CRISPR/Cas target sequence and the PAM would be integrated into the genome.
  • a wild type CRISPR/Cas protein e.g., Cas9 protein
  • Cas9 protein normally has nuclease activity that cleaves a target nucleic acid (e.g., a double stranded DNA (dsDNA)) at a target site defined by the region of complementarity between the guide sequence of the guide RNA and the target nucleic acid.
  • site-specific targeting to the target nucleic acid occurs at locations determined by both (i) base-pairing complementarity between the guide nucleic acid and the target nucleic acid; and (ii) a short motif referred to as the “protospacer adjacent motif” (PAM) in the target nucleic acid.
  • PAM protospacer adjacent motif
  • a Cas9 protein binds to (in some cases cleaves) a dsDNA target nucleic acid
  • the PAM sequence that is recognized (bound) by the Cas9 polypeptide is present on the non-complementary strand (the strand that does not hybridize with the targeting segment of the guide nucleic acid) of the target DNA.
  • CRISRPR/Cas e.g., Cas9 proteins from different species can have different PAM sequence requirements.
  • a nucleic acid that binds to a class 2 CRISPR/Cas endonuclease e.g., a Cas9 protein; a type V or type VI CRISPR/Cas protein; a Cpf1 protein; etc.
  • a guide RNA or “CRISPR/Cas guide nucleic acid” or “CRISPR/Cas guide RNA.”
  • a guide RNA provides target specificity to the complex (the RNP complex) by including a targeting segment, which includes a guide sequence (also referred to herein as a targeting sequence), which is a nucleotide sequence that is complementary to a sequence of a target nucleic acid.
  • a cell includes a plurality of such cells and reference to “the regulatory T cell-like cells” includes reference to one or more regulatory T cell-like cells and equivalents thereof, e.g. CD4 edFOXP3 cells, Treg-like cells, or engineered Tregs, known to those skilled in the art, and so forth.
  • the regulatory T cell-like cells includes reference to one or more regulatory T cell-like cells and equivalents thereof, e.g. CD4 edFOXP3 cells, Treg-like cells, or engineered Tregs, known to those skilled in the art, and so forth.
  • Tolerogenic means capable of suppressing or down-modulating an adaptive or innate immunological response.
  • biological sample encompasses a clinical sample.
  • the types of “biological samples” include, but are not limited to: tissue obtained by surgical resection, tissue obtained by biopsy, cells in culture, cell supernatants, cell lysates, tissue samples, organs, bone marrow, blood, plasma, serum, fine needle aspirate, lymph node aspirate, cystic aspirate, a paracentesis sample, a thoracentesis sample, and the like.
  • the terms “obtained” or “obtaining” as used herein can also include the physical extraction or isolation of a biological sample (e.g., comprising HSPC, lymphoid progenitors, CD4 + T lymphocytes) from a subject.
  • a biological sample comprising hematopoietic cells can be isolated from a subject (and thus “obtained”) by the same person or same entity that subsequently isolates HSPC, CD4 + T lymphocytes, etc. from the sample and produces CD4 edF ox P3 T cells (gene edited with CRISPR/Cas9 and FOXP3 homology donor vectors) from the original unmodified cells in the sample.
  • the step of obtaining does not comprise the step of isolating a biological sample.
  • the step of obtaining comprises the step of isolating a biological sample (e.g., a pre-treatment biological sample, a post-treatment biological sample, etc.).
  • a biological sample e.g., a pre-treatment biological sample, a post-treatment biological sample, etc.
  • Methods and protocols for isolating various biological samples e.g., a blood sample, a biopsy sample, an aspirate, etc. will be known to one of ordinary skill in the art and any convenient method may be used to isolate a biological sample.
  • substantially purified generally refers to isolation of a component of a sample (e.g., cell or substance), such that the component comprises the majority percent of the sample in which it resides.
  • a substantially purified component comprises at least 70%, preferably at least 80%-85%, more preferably at least 90-99% of the sample.
  • treatment used herein to generally refer to obtaining a desired pharmacologic and/or physiologic effect.
  • the effect can be prophylactic in terms of completely or partially preventing a disease or symptom(s) thereof and/or may be therapeutic in terms of a partial or complete stabilization or cure for a disease and/or adverse effect attributable to the disease.
  • treatment encompasses any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease and/or symptom(s) from occurring in a subject who may be predisposed to the disease or symptom but has not yet been diagnosed as having it; (b) inhibiting the disease and/or symptom(s), i.e., arresting their development; or (c) relieving the disease symptom(s), i.e., causing regression of the disease and/or symptom(s).
  • Those in need of treatment include those already inflicted as well as those in which prevention is desired (e.g., those with increased susceptibility to an autoimmune disease, etc.)
  • a therapeutic treatment is one in which the subject is inflicted prior to administration and a prophylactic treatment is one in which the subject is not inflicted prior to administration.
  • the subject has an increased likelihood of becoming inflicted or is suspected of being inflicted prior to treatment.
  • the subject is suspected of having an increased likelihood of becoming inflicted.
  • “Pharmaceutically acceptable excipient or carrier” refers to an excipient that may optionally be included in the compositions of the invention and that causes no significant adverse toxicological effects to the patient.
  • “Pharmaceutically acceptable salt” includes, but is not limited to, amino acid salts, salts prepared with inorganic acids, such as chloride, sulfate, phosphate, diphosphate, bromide, and nitrate salts, or salts prepared from the corresponding inorganic acid form of any of the preceding, e.g., hydrochloride, etc., or salts prepared with an organic acid, such as malate, maleate, fumarate, tartrate, succinate, ethylsuccinate, citrate, acetate, lactate, methanesulfonate, benzoate, ascorbate, para-toluenesulfonate, palmoate, salicylate and stearate, as well as estolate, gluceptate and lactobionate salts.
  • salts containing pharmaceutically acceptable cations include, but are not limited to, sodium, potassium, calcium, aluminum, lithium, and ammonium (including substituted ammonium).
  • an "effective amount" of a composition comprising HSPC edF0XP3 or CD4 edFOXP3 T cells is an amount sufficient to safely effect beneficial or desired results, such as an amount that suppresses activation and proliferation of effector T cells and increases immune tolerance.
  • An effective amount can be administered in one or more administrations, applications, or dosages.
  • a composition comprising HSPC edF0XP3 or CD4 edFOXP3 T cells is intended an amount that, when administered as described herein, brings about a positive therapeutic response, such as improved recovery from an inflammatory condition such as, but not limited to, an autoimmune manifestation, an allergy, graft-versus-host disease, and transplant rejection. Improved recovery may include a reduction in inflammation, pain, or autoimmune-induced tissue damage, or better graft tolerance and prolonged survival of transplanted cells, tissue or organs. Additionally, a therapeutically effective dose or amount may compensate for functional (e.g., IPEX syndrome) or quantitative Treg-deficiency and reduce the need for immunosuppressive or anti-inflammatory drugs.
  • an effective unit dose may be 10 6 cells /kg, 3 c 10 6 cells /kg, 10 7 cells /kg, 10 8 cells /kg, 10 9 /kg, or more.
  • isolated is meant, when referring to a polypeptide, that the indicated molecule is separate and discrete from the whole organism with which the molecule is found in nature or is present in the substantial absence of other biological macro molecules of the same type.
  • isolated with respect to a polynucleotide is a nucleic acid molecule devoid, in whole or part, of sequences normally associated with it in nature; or a sequence, as it exists in nature, but having heterologous sequences in association therewith; or a molecule disassociated from the chromosome.
  • isolated when referring to a cell, is a cell that is separate and discrete from the whole organism with which the cell is found in nature.
  • substantially purified generally refers to isolation of a substance (compound, drug, polynucleotide, protein, polypeptide) such that the substance comprises the majority percent of the sample in which it resides.
  • a substantially purified component comprises 50%, preferably 80%-85%, more preferably 90-95% of the sample.
  • Techniques for purifying substances of interest are well-known in the art and include, for example, ion-exchange chromatography, affinity chromatography and sedimentation according to density.
  • the terms “recipient”, “individual”, “subject”, “host”, and “patient”, are used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans.
  • mammal for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, etc. Preferably, the mammal is human.
  • unit dosage form refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of the agents calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle.
  • the specifications for the unit dosage forms for use in the present invention depend on the particular compound employed and the effect to be achieved, the pharmacodynamics associated with each compound in the host, and the like.
  • Recombinant as used herein to describe a nucleic acid molecule means a polynucleotide of genomic, cDNA, viral, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation, is not associated with all or a portion of the polynucleotide with which it is associated in nature.
  • the term "recombinant” as used with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide.
  • the gene of interest is cloned and then expressed in transformed organisms, as described further below. The host organism expresses the foreign gene to produce the protein under expression conditions.
  • transformation refers to the insertion of an exogenous polynucleotide into a host cell, irrespective of the method used for the insertion.
  • Recombinant host cells refer to cells which can be, or have been, used as recipients for recombinant vector or other transferred DNA, and include the original progeny of the original cell which has been transfected.
  • operably linked refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function.
  • a given promoter operably linked to a coding sequence is capable of effecting the expression of the coding sequence when the proper enzymes are present.
  • Expression is meant to include the transcription of mRNA from a DNA or RNA template and can further include translation of a protein from an mRNA template.
  • the promoter need not be contiguous with the coding sequence, so long as it functions to direct the expression thereof.
  • intervening untranslated yet transcribed sequences can be present between the promoter sequence and the coding sequence and the promoter sequence can still be considered “operably linked" to the coding sequence.
  • a “vector” is capable of transferring nucleic acid sequences to target cells (e.g., viral vectors, non-viral vectors, particulate carriers, and liposomes).
  • target cells e.g., viral vectors, non-viral vectors, particulate carriers, and liposomes.
  • vector construct means any nucleic acid construct capable of directing the expression of a nucleic acid of interest and which can transfer nucleic acid sequences to target cells.
  • the term includes cloning and expression vehicles, as well as viral vectors.
  • variant refers to biologically active derivatives of the reference molecule that retain desired activity.
  • variant refers to molecules having a native sequence and structure with one or more additions, substitutions (generally conservative in nature) and/or deletions, relative to the native molecule, so long as the modifications do not destroy biological activity and which are "substantially homologous" to the reference molecule.
  • sequences of such variants will have a high degree of sequence homology to the reference sequence, e.g., sequence homology of more than 50%, generally more than 60%-70%, even more particularly 80%-85% or more, such as at least 90%-95% or more, when the two sequences are aligned.
  • Gene transfer or “gene delivery” refers to methods or systems for reliably inserting DNA or RNA of interest into a host cell. Such methods can result in transient expression of non- integrated transferred DNA, extrachromosomal replication and expression of transferred replicons (e.g., episomes), or integration of transferred genetic material into the genomic DNA of host cells.
  • transferred replicons e.g., episomes
  • a polynucleotide "derived from" a designated sequence refers to a polynucleotide sequence which comprises a contiguous sequence of approximately at least about 6 nucleotides, preferably at least about 8 nucleotides, more preferably at least about 10-12 nucleotides, and even more preferably at least about 15-20 nucleotides corresponding, i.e., identical or complementary to, a region of the designated nucleotide sequence.
  • the derived polynucleotide will not necessarily be derived physically from the nucleotide sequence of interest, but may be generated in any manner, including, but not limited to, chemical synthesis, replication, reverse transcription or transcription, which is based on the information provided by the sequence of bases in the region(s) from which the polynucleotide is derived. As such, it may represent either a sense or an antisense orientation of the original polynucleotide.
  • Homology-directed repair is a mechanism in cells to repair double-stranded and single stranded DNA breaks.
  • Homology-directed repair includes homologous recombination (HR) and single-strand annealing (SSA) (Lieber. 2010 Annu. Rev. Biochem. 79:181-211).
  • HR homologous recombination
  • SSA single-strand annealing
  • Other forms of HDR include single-stranded annealing (SSA) and breakage-induced replication, and these require shorter sequence homology relative to HR.
  • CRISPR is used to edit pre-existing FOXP3 mutants in order to replace with a desired version/variant of FOXP3.
  • CRISPR based genome editing methods provide advantages over traditional lentiviral methods of gene addition. Advantages include but are not limited to, increased breath of the cells types that can be transformed, allows for FOXP3 expression to be controlled by the endogenous FOXP3 promoter, allows locus specific replacement with correction of many different mutation types, etc.
  • compositions, methods, and kits are provided for producing and using engineered hematopoietic cells capable of expressing FOXP3.
  • FOXP3 is a transcription factor essential for the function of natural Tregs in maintenance of immune tolerance and normal Teff function.
  • CRISPR/Cas9-mediated gene editing of FOXP3 in CD4 + T lymphocytes endows cells with Treg- like characteristics, including the ability to suppress immune responses of effector T cells and other immune cells.
  • CD4 edFOXP3 Treg cells are useful for increasing immune tolerance to antigens in a subject such as alloantigens, autoantigens, and allergens.
  • pharmaceutical compositions comprising such engineered CD4 edFOXP3 T cells, or compositions of stem and progenitor cells that can give rise to CD4 edFOXP3 T cells.
  • a CRISPR/Cas9 vector comprising a CRISPR/Cas9 system cuts the endogenous FOXP3 gene at the target site of the sgRNA. After cutting, the FOXP3 homology donor vector then replaces the endogenous copy of FOXP3 with the desired version/variant of FOXP3 contained within the FOXP3 homology donor vector using homology directed repair in a hematopoietic cell, converting them into gene edited cells.
  • nucleic acids encoding the forkhead box protein 3 (FOXP3) transcription factor can be inserted into the FOXP3 homology donor vector to create a vector capable of replacing the endogenous copy of FOXP3 with a desired version/variant following CRISPR/Cas9 cutting/editing.
  • FOXP3 forkhead box protein 3
  • the recombinant FOXP3 homology donor vector comprises: a) a 5’ homology arm; b) a polynucleotide encoding forkhead box protein 3 (FOXP3) or a variant thereof; c) a polyadenylation sequence d) a phosphoglycerate kinase 1 (PGK) promoter, wherein the PGK promoter is operably linked to the polynucleotide encoding a cell surface marker; e) a polynucleotide encoding a cell surface marker for in vitro selection and in vivo tracking of cells transduced with the vector; and f) a 3’ homology arm.
  • FOXP3 forkhead box protein 3
  • PGK phosphoglycerate kinase 1
  • the cell surface marker is a truncated nerve growth factor receptor (tNGFR).
  • the recombinant FOXP3 homology donor vector comprises the nucleotide sequence of SEQ ID NO:2 or a sequence having at least about 80-100% sequence identity thereto, including any percent identity within this range, such as 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, wherein the recombinant FOXP3 homology donor vector is capable of generating a Treg-like cell by transfection of a CD4+ T lymphocyte.
  • constructs to produce FOXP3 can be empirically determined, for example, by using a real-time RT-PCR assay of FOXP3 mRNA levels or a Western Blot assay of FOXP3 protein levels. Additionally, the ability of the CRISPR/Cas9 and FOXP3 homology donor vector to confer physiologic Teff or Treg characteristics on CD4 + T lymphocytes can be evaluated with a proliferation or a suppression assay, respectively, in vitro (see Examples).
  • FOXP3 nucleic acid and protein sequences may be derived from any source.
  • a number of FOXP3 nucleic acid and protein sequences are known.
  • a representative example of a human FOXP3 sequences is presented in SEQ ID NO:1 and SEQ ID NO:2, and additional representative sequences including various isoforms of the FOXP3 transcription factor are listed in the National Center for Biotechnology Information (NCBI) database. See, for example, NCBI entries: Accession Nos.
  • any of these sequences or a variant thereof comprising a sequence having at least about 80-100% sequence identity thereto, including any percent identity within this range, such as 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, can be used to construct a FOXP3 homology donor construct, wherein the expressed variant FOXP3 retains biological activity, including transcription factor activity and the ability to convert CD4 + T lymphocytes into CD4 edFOXP3 T cells.
  • the hematopoietic cells can optionally be purified before or after gene editing by any method known in the art, including, but not limited to, density gradient centrifugation (e.g., Ficoll Hypaque, percoll, iodoxanol and sodium metrizoate), immunoselection (positive selection or negative selection for surface markers) with immunomagnetic beads or immunoaffinity columns, or fluorescence-activated cell sorting (FACS).
  • density gradient centrifugation e.g., Ficoll Hypaque, percoll, iodoxanol and sodium metrizoate
  • immunoselection positive selection or negative selection for surface markers
  • immunomagnetic beads or immunoaffinity columns e.g., immunomagnetic beads or immunoaffinity columns
  • FACS fluorescence-activated cell sorting
  • CD4 + T lymphocytes or CD34+ HSPC can be isolated from apheresis products by immunomagnetic CD4 + cell selection, cultured in the presence of IL-2 and IL-7, then transfected or transduced with a FOXP3 homology donor vector, followed by immunoselection for the cell surface marker (e.g., truncated NGFR) expressed by the recombinant FOXP3 homology donor vector to separate gene edited cells from non-gene edited cells (see, Examples).
  • a FOXP3 homology donor vector e.g., truncated NGFR
  • Hematopoietic stem cells can be obtained by harvesting from bone marrow, from peripheral blood or cord blood. Bone marrow is generally aspirated from the posterior iliac crests while the donor is under either regional or general anesthesia. Additional bone marrow can be obtained from the anterior iliac crest. A dose of 1 X 10 8 and 2 X 10 8 marrow mononuclear cells per kilogram is generally considered desirable to establish engraftment in autologous and allogeneic marrow transplants, respectively. Bone marrow can be primed with granulocyte colony-stimulating factor (G-CSF; filgrastim [Neupogen]) to increase the stem cell count.
  • G-CSF granulocyte colony-stimulating factor
  • G-CSF cytokines
  • GM-CSF GM-CSF
  • the stem cells are optionally, although not necessarily, purified.
  • Abundant reports explore various methods for purification of stem cells and subsequent engraftment, including flow cytometry; an isolex system (Klein et al. (2001) Bone Marrow Transplant. 28(11 ):1023-9; Prince et al. (2002) Cytotherapy 4(2):137-45); immunomagnetic separation (Prince et al. (2002) Cytotherapy 4(2):147-55; Handgretinger et al. (2002) Bone Marrow Transplant. 29(9):731-6; Chou et al. (2005) Breast Cancer. 12(3) :178-88); and the like.
  • Each of these references is herein specifically incorporated by reference, particularly with respect to procedures, cell compositions and doses for hematopoietic stem cell transplantation.
  • the cells which are employed may be fresh, frozen, or have been subject to prior culture. They may be fetal, neonate, adult, etc. Hematopoietic stem cells may be obtained from fetal liver, bone marrow, cord blood, blood, particularly G-CSF or GM-CSF mobilized peripheral blood, or any other conventional source. Cells for engraftment are optionally isolated from other cells, where the manner in which the stem cells are separated from other cells of the hematopoietic or other lineage is not critical to this invention. If desired, a substantially homogeneous population of stem or progenitor cells may be obtained by selective isolation of cells free of markers associated with differentiated cells, while displaying epitopic characteristics associated with the stem cells.
  • the ability of the resulting engineered Teff or Treg CD4 edFOXP3 cells to respond to activation or to suppress proliferation and activation of effector T cells and other immune cells can be assayed by methods well known in the art including, for example, without limitation, performing an in vitro suppression assay or 3 H-thymidine assay that measures suppression of T cell proliferation by CD4 edFOXP3 T cells, or a flow cytometry-based suppression assay that measures suppression of proliferation and cytokine production in subpopulations of T cells and other immune cells (see, e.g., Thornton et al. (1998) J. Exp. Med. 1998. 188:287-296, Schneider et al. (2011) Methods Mol. Biol.
  • Methods are provided for restoring a multilineage T cell compartment in individuals with mutated FOXP3, including, for example, IPEX.
  • the methods described herein are also useful for treating various immune conditions and disorders benefitting from increased immunological tolerance, such as inflammatory conditions including for example, without limitation, Treg deficiency, autoimmune disorders, allergies, graft-versus-host disease, and organ or tissue transplantation.
  • polyclonal CD4 edFOXP3 T cells which may be derived in vivo from transplanted HSPC edF0XP3 , comprising a plurality of different T cell receptors, are used for immunosuppression and promoting immune tolerance generally.
  • CD4 edF ox P3 T cells comprising a T cell receptor specific for an antigen of interest are used to dampen adaptive antigen-specific immune responses to the antigen of interest selectively.
  • the infusion of gene edited cells is a relatively simple process that is performed at the bedside.
  • the gene edited cells are infused through a central vein over a period of several hours.
  • Autologous products are frequently cryopreserved; if so they are thawed at the bedside and infused rapidly over a period of several minutes.
  • the dose of HSC is at least about 10 5 CD34 + cells/kg body weight, at least about 0.5 x 10 6 , at least about 10 6 , and up to about 2.5 x 10 6 , 5 x 10 6 , 7.5 x 10 6 , 10 7 CD34 + cells/kg body weight.
  • CD34 + cells For positive selection of CD34 + cells, commercial instruments can be employed to remove the desired cells, using solid-phase, anti-CD34 monoclonal antibodies. With negative selection, monoclonal antibodies can be used to remove undesired cells, leaving stem cells in the graft.
  • Treg deficiency and autoimmune and other inflammatory conditions that may be treated with engineered HSPC or CD4 edFOXP3 T cells by the methods described herein include, but are not limited to, immune dysregulation polyendocrinopathy enteropathy X-linked (IPEX) syndrome.
  • Other conditions associated with autoimmunity and undesirable inflammation include, for example, genetic conditions with Treg impairment and certain degree of Teff abnormality could be treated with similar approaches using other genes instead of FOXP3 (see Cepika AM, JACI 2019).
  • Treatment of primates, more particularly humans is of interest, but other mammals may also benefit from treatment, particularly domestic animals such as equine, bovine, ovine, feline, canine, murine, lagomorpha, and the like.
  • compositions can be prepared by formulating the FOXP3 edited hematopoietic cells into dosage forms by known pharmaceutical methods.
  • a pharmaceutical composition comprising FOXP3 edited hematopoietic cells can be formulated for parenteral administration, as liquids, suspensions, emulsions, and injections (such as venous injections, drip injections, and the like).
  • the FOXP3 edited hematopoietic cells can be combined as appropriate, with pharmaceutically acceptable carriers or media, in particular, sterile water and physiological saline, vegetable oils, resolvents, bases, emulsifiers, suspending agents, surfactants, stabilizers, vehicles, antiseptics, binders, diluents, tonicity agents, soothing agents, bulking agents, disintegrants, buffering agents, coating agents, lubricants, coloring agents, solution adjuvants, or other additives.
  • pharmaceutically acceptable carriers or media in particular, sterile water and physiological saline, vegetable oils, resolvents, bases, emulsifiers, suspending agents, surfactants, stabilizers, vehicles, antiseptics, binders, diluents, tonicity agents, soothing agents, bulking agents, disintegrants, buffering agents, coating agents, lubricants, coloring agents, solution adjuvants, or other additives.
  • pharmaceutically acceptable carriers or media in particular,
  • the subject who receives the FOXP3 edited hematopoietic cells is also the subject from whom the original, unmodified cells are harvested or obtained, which provides the advantage that the donated cells are autologous.
  • FOXP3 edited hematopoietic cells can be obtained from another subject (i.e., donor), a culture of cells from a donor, or from established cell culture lines.
  • FOXP3 edited hematopoietic cells may be obtained from the same species than the subject to be treated, and more preferably of the same immunological profile as the subject.
  • Such cells can be obtained, for example, from a biological sample comprising FOXP3 edited hematopoietic cells from a close relative or matched donor, and the FOXP3 edited hematopoietic cells that are produced (i.e., gene editing with a CRISPR/Cas 9 vector and a FOXP3 homology donor vector) can be administered to a subject in need of treatment for an inflammatory condition.
  • FOXP3 edited hematopoietic cells i.e., gene editing with a CRISPR/Cas 9 vector and a FOXP3 homology donor vector
  • the FOXP3 edited hematopoietic cells that are administered to a subject are derived from autologous or allogeneic cells.
  • the patients or subjects who donate or receive the cells are typically mammalian, and usually human. However, this need not always be the case, as veterinary applications are also contemplated.
  • At least one therapeutically effective cycle of treatment with FOXP3 edited hematopoietic cells will be administered to a subject for treatment of an inflammatory condition.
  • FOXP3 edited hematopoietic cells i.e., HSPC, lymphoid progenitors or CD4 + T lymphocytes gene edited with a CRISPR/Cas9 vector and a FOXP3 homology donor vector
  • a therapeutically effective dose or amount of a composition comprising FOXP3 edited hematopoietic cells is intended an amount that, when administered as described herein, brings about a positive therapeutic response, such as improved recovery from an inflammatory condition benefitting from increased immunological tolerance, such as an autoimmune disorder, an allergy, graft-versus-host disease, or a tissue transplant.
  • Improved recovery may include a reduction in inflammation, pain, or autoimmune-induced tissue damage, decreased allergic response, or prolonged survival of transplanted tissue or organs. Additionally, a therapeutically effective dose or amount may compensate for Treg-deficiency (e.g., IPEX syndrome) and reduce the need for immunosuppressive or anti-inflammatory drugs.
  • Treg-deficiency e.g., IPEX syndrome
  • compositions comprising FOXP3 edited hematopoietic cells and/or one or more other therapeutic agents, such as other drugs for treating immune diseases or conditions, or other medications will be administered.
  • the compositions of the present invention are typically, although not necessarily, administered via injection (subcutaneously, intravenously, intra-arterially, or intramuscularly), by infusion, or locally. Additional modes of administration are also contemplated, such as intraperitoneal, intrathecal, intralymphatic, intravascular, intralesion, transdermal, and so forth.
  • the pharmaceutical compositions comprising FOXP3 edited hematopoietic cells and other agents may be administered using the same or different routes of administration in accordance with any medically acceptable method known in the art.
  • the pharmaceutical compositions comprising FOXP3 edited hematopoietic cells are administered prophylactically, e.g., to prevent Treg deficiency, etc.
  • prophylactic uses will be of particular value for subjects who have a disease or a genetic predisposition to developing an inflammatory condition, such as an autoimmune disease, inflammation, or allergy.
  • FOXP3 edited hematopoietic cells may be administered to a patient with an autoimmune disease to prevent a disease flare, or in IPEX patients with mixed donor chimerism and disease relapse.
  • compositions comprising FOXP3 edited hematopoietic cells can effectively treat.
  • the actual dose and number of doses to be administered will vary depending upon the age, weight, and general condition of the subject as well as the severity of the condition being treated, the judgment of the health care professional, and conjugate being administered.
  • Therapeutically effective amounts can be determined by those skilled in the art, and will be adjusted to the particular requirements of each particular case.
  • compositions comprising FOXP3 edited hematopoietic cells, prepared as described herein (again, preferably provided as part of a pharmaceutical preparation), can be administered alone or in combination with one or more other therapeutic agents for treating an immune disease or condition.
  • Antibody conditioning may be used, or myeloablative conditioning as known in the art.
  • Individuals nay be treated with combination therapies with other medications used to treat a particular condition or disease according to a variety of dosing schedules depending on the judgment of the clinician, needs of the patient, and so forth. The specific dosing schedule will be known by those of ordinary skill in the art or can be determined experimentally using routine methods.
  • Exemplary dosing schedules include, without limitation, administration five times a day, four times a day, three times a day, twice daily, once daily, three times weekly, twice weekly, once weekly, twice monthly, once monthly, and any combination thereof.
  • Preferred compositions are those requiring dosing no more than once a day.
  • compositions comprising FOXP3 edited hematopoietic cells can be administered prior to, concurrent with, or subsequent to other agents. If provided at the same time as other agents, the FOXP3 edited hematopoietic cells can be provided in the same or in a different composition. Thus, the FOXP3 edited hematopoietic cells and one or more other agents can be presented to the individual by way of concurrent therapy.
  • concurrent therapy is intended administration to a subject such that the therapeutic effect of the combination of the substances is caused in the subject undergoing therapy.
  • concurrent therapy may be achieved by administering a dose of a pharmaceutical composition comprising FOXP3 edited hematopoietic cells and a dose of a pharmaceutical composition comprising at least one other agent, such as a drug for treating an immune disease or condition, which in combination comprise a therapeutically effective dose, according to a particular dosing regimen.
  • the FOXP3 edited hematopoietic cells and one or more other therapeutic agents can be administered in at least one therapeutic dose.
  • Administration of the separate pharmaceutical compositions can be performed simultaneously or at different times (i.e., sequentially, in either order, on the same day, or on different days), as long as the therapeutic effect of the combination of these substances is caused in the subject undergoing therapy.
  • compositions described herein may be included in a kit.
  • hematopoietic edFOXP3 cells i.e., gene corrected CD4 + T lymphocytes, gene corrected HSPC, etc.
  • a CRISPR/Cas9 vector and a FOXP3 homology donor vector, as described herein, for expression of FOXP3 hematopoietic cells to produce CD4 edFOXP3 T cells may be included in the kit.
  • untransduced hematopoietic cells are provided with the CRISPR/Cas9 RNP complex and the FOXP3 homology donor vectors separate.
  • the kit may also comprise nucleotransfection agents, agents for purification of cells (e.g., microbeads for selection of transfected cells having the NGFR surface marker), agents for maintaining or culturing cells, such as media, and optionally one or more other factors, such as cytokines (e.g., IL-2), growth factors, antibiotics, and the like.
  • agents for purification of cells e.g., microbeads for selection of transfected cells having the NGFR surface marker
  • agents for maintaining or culturing cells such as media
  • optionally one or more other factors such as cytokines (e.g., IL-2), growth factors, antibiotics, and the like.
  • kits generally will comprise, in suitable means, distinct containers for each individual reagent or solution.
  • the kit may comprise one or more containers holding the hematopoietic cells and/or CRISPR/Cas9 vector and FOXP3 homology donor vectors, and other agents.
  • Suitable containers for the compositions include, for example, bottles, vials, syringes, and test tubes.
  • Containers can be formed from a variety of materials, including glass or plastic.
  • a container may have a sterile access port (for example, the container may be a vial having a stopper pierceable by a hypodermic injection needle).
  • the kit can further comprise a container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution, or dextrose solution. It can also contain other materials useful to the end-user, including other pharmaceutically acceptable formulating solutions such as buffers, diluents, filters, needles, and syringes or other delivery devices.
  • a pharmaceutically-acceptable buffer such as phosphate-buffered saline, Ringer's solution, or dextrose solution.
  • the delivery device may be pre-filled with the compositions.
  • the kit can also comprise a package insert containing written instructions for methods of treating inflammatory conditions with the cells, as described herein.
  • the package insert can be an unapproved draft package insert or can be a package insert approved by the Food and Drug Administration (FDA) or other regulatory body.
  • FDA Food and Drug Administration
  • the kit comprises a CRISPR/Cas9 vector and a FOXP3 homology donor vector comprising the components arranged as depicted in the vector map shown in FIG. 1 A.
  • the kit comprises a recombinant FOXP3 homology donor vector , comprising a coding sequence for FOXP3, usually a full-length coding sequence.
  • the coding sequence may be a cDNA, or may comprise one or more introns.
  • the coding sequence can be modified, or diverged, to incorporate synonymous mutations at the nucleotide level according to the redundant codon usage system, to prevent premature recombination while still encoding for a wild-type protein.
  • the FOXP3 sequence encodes a functional, wild-type FOXP3 protein, although for research purposes a mutated form may be encoded.
  • the FOXP3 protein may be one or both of the FOXP3 isoforms FOXP 1 (SEQ ID NO:1) and FOXP ⁇ 2 (SEQ ID NO:2).
  • the FOXP3 coding sequence is generally not linked to a promoter in the vector, and is expressed in the cell by the native FOXP3 promoter.
  • the FOXP3 coding sequence may be operably linked to a polyadenylation sequence, including without limitation BGH polyadenylation signal.
  • the homology vector optionally comprises a marker sequence, including without limitation a truncated nerve growth factor receptor (tNGFR) operably linked to a promoter, e.g. the phosphoglycerate kinase 1 (PGK) promoter.
  • tNGFR truncated nerve growth factor receptor
  • PGK phosphoglycerate kinase 1
  • the homology donor vector further comprises a 5’ and 3’ arm of homology to the chromosomal site; where the homology arms may be centered on the cut site of the sgRNA.
  • the recombinant FOXP3 homology donor vector comprises the nucleotide sequence of SEQ ID NO:3 or SEQ ID NO:4, or a sequence having at least about 80- 100% sequence identity thereto, including any percent identity within this range, such as 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, wherein the recombinant FOXP3 homology donor vector is capable of gene correcting a mutated FOXP3 sequence in a hematopoietic cell of interest.. [00112] It will be apparent to one of ordinary skill in the art that various changes and modifications can be made without departing from the spirit or scope of the invention.
  • the prototypical genetic autoimmune disease is immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome, which is a severe, and often fatal, pediatric disease with limited treatment options.
  • IPEX syndrome is caused by mutations in the forkhead box protein 3 ( FOXP3 ) gene, which is a critical transcription factor required for thymic- derived regulatory T cell (Treg) and CD4+ effector T (Teff) cell function.
  • FOXP3 forkhead box protein 3
  • Treg thymic- derived regulatory T cell
  • Teff CD4+ effector T
  • IPEX is an ideal candidate for a gene therapy approach whereby patient hematopoietic stem and progenitor (HSPC) cells or T cells are gene corrected ex vivo and reinfused in the patient.
  • HSPC patient hematopoietic stem and progenitor
  • the FOXP3 locus is efficiently and precisely edited by CRISPR in human HSPCs and T cells.
  • CRISPR-system targeting the FOXP3 gene downstream of the translation start codon in exon 1 and a corresponding HDR donor containing FOXP3 cDNA (Fig. 1A).
  • the donor construct was designed to insert a codon diverged FOXP3 cDNA and restore wild-type FOXP3 protein expression in patient cells with diverse and scattered FOXP3 mutations.
  • the gene replacement donor template was also designed to knock-in a marker gene, the truncated nerve growth factor receptor (tNGFR), which is used clinically as a surface marker for selection and tracking of genetically engineered cells.
  • tNGFR truncated nerve growth factor receptor
  • FOXP3FL full length FOXP3 cDNA construct
  • FOXP3dE2 FOXP3dE2
  • FOXP3KO FOXP3 knockout construct
  • sgRNAs FOXP3 CRISPR single-guide RNA
  • sgRNA 2 was selected due to a combination of its on-target activity, safe predicted off-target profile, location in the coding sequence of the gene, and proximity to the start codon.
  • Testing of the alternative constructs, FOXP3dE2 and FOXP3KO, in HSPCs revealed average HDR rates of 26 ⁇ 9% and 23 ⁇ 5% respectively (Fig. 8B).
  • the CRISPR system enables specific FOXP3 gene editing in HSPCs.
  • bioinformatic prediction a double strand break (DSB) capture assay
  • NGS next generation sequencing
  • 58 potential off- target sites were predicted by bioinformatic in silico prediction using the CRISPR Search with Mismatches, Insertions and/or Deletions (COSMID) tool (25), of which 96% were in non-coding regions of the genome (Fig. 8E, Table 8).
  • COSMID CRISPR Search with Mismatches, Insertions and/or Deletions
  • Ten off-target sites were identified using DSB capture by GUIDE-seq (26) in the U20S cell line, of which seven were also predicted by in silico analysis (Fig. 2D and Fig. 8F).
  • the 61 sites predicted by the combination of COSMID and GUIDE-seq were then evaluated in FOXP3 edited HSPCs by NGS.
  • Four sites were validated by NGS, three of which were identified by all three methods.
  • the four sites identified as off-targets were ranked as 1 , 3, 4, and 14 by the COSMID algorithm.
  • none of the validated sites had more than three mismatches.
  • all off-target sites had less than 2% targeting (Fig. 2E).
  • FOXP3 edited Tregs express persistent FOXP3 protein and display Treg phenotype and function.
  • FOXP3 cDNA insertion strategy in Tregs, the major cell type that expresses FOXP3 and is implicated in IPEX syndrome.
  • FOXP3 expression in MT-2 cells was tested that FOXP3FL edited cells expressed FOXP3 protein, but at a lower level compared to unmodified MT-2 cells (Fig. 3A and Fig. 10A).
  • FOXP3FLco codon optimized FOXP3 cDNA construct
  • FOXP3co + WPRE or FOXP3FLcoW a codon optimized FOXP3 cDNA construct
  • FOXP3co + WPRE or FOXP3FLcoW a woodchuck hepatitis virus posttranscriptional regulatory element
  • FOXP3dE2 Tregs we tested the suppressive function of FOXP3dE2 Tregs and found them to perform comparably to FOXP3FL and FOXP3FLcoW edited Tregs (Fig. 11 B-C).
  • FOXP3FLcoW edited Teff cells that were cultured and edited in parallel were tested and found to lack suppressive function (Fig. 11C-D).
  • FOXP3FLcoW construct Given the ability to suppress Teff proliferation and the relatively higher FOXP3 expression, we selected the FOXP3FLcoW construct for subsequent functional testing.
  • FOXP3 cDNA knock-in Tregs displayed suppressive function that overlapped with lower normal range of suppressive function observed in WT Tregs from different donors, suggesting that editing might be sufficient to restore suppressive capacity to non-functional Tregs
  • FOXP3 gene editing permits physiologically regulated FOXP3 expression and preserved function in Teff cells. Because Teff cells transiently express FOXP3 upon TCR activation, we monitored FOXP3 protein expression in FOXP3 edited Teff cells by flow cytometry over a two week time course after activation. In non-activated cells, a low level of background FOXP3 expression was observed, likely due to the pre-editing activation and culturing (Fig. 4A). Upon TCR- mediated re-activation, FOXP3 expression in both edited cells and controls was induced and nearly doubled by day three before gradually returning to baseline (Fig. 4A).
  • CRISPR-based editing restores functional FOXP3 expression to IPEX patient Tregs and Teff cells.
  • IPEX patient Tregs and Teff cells We obtained cells from six IPEX patients including two sets of brothers with identical pathologic mutations (mutation locations depicted in Fig. 5A; see Table 9 for patient information). Mutations ranged from point mutations to complete abrogation of gene expression.
  • FOXP3FLcoW edited and control HSPCs (WT unmodified and WT mock) were injected into the liver of three to four day old neonatal immunodeficient mice, and engraftment was monitored over a 14 week time course (Fig.6B).
  • the NSG-SGM3 strain of mice was selected for engraftment studies due to their expression of several human cytokines and their reported higher proportion of FOXP3+ Tregs relative to standard NSG mice (28).
  • edited HSPCs Prior to injection, edited HSPCs were phenotyped by flow cytometry for purity (%CD34+) and markers of hematopoietic progenitor subsets (Fig. 13A).
  • mice Gene edited and control HSPCs from three cord blood donors were injected, without prior enrichment for tNGFR, into three corresponding litters of mice. A total of 27 mice were injected, including ten FOXP3FLcoW edited, nine WT unmodified, and eight WT mock conditions. The overall survival of the mice over the course of the study was comparable among conditions (Fig. 13B). Human cell engraftment, determined by flow cytometry analysis of hCD45+ expression, steadily increased over time in the peripheral blood of the mice and was found to be comparable among conditions with no statistically significant differences (Fig. 6C).
  • hematopoietic lineages were analyzed by flow cytometry and among the edited conditions, the cells were sub-gated into tNGFR+ and tNGFR- fractions for comparison (Fig. 6D- E).
  • Fig. 6D- E The hematopoietic lineages were analyzed by flow cytometry and among the edited conditions, the cells were sub-gated into tNGFR+ and tNGFR- fractions for comparison.
  • Fig. 6D- E The hematopoietic lineages were analyzed by flow cytometry and among the edited conditions, the cells were sub-gated into tNGFR+ and tNGFR- fractions for comparison (Fig. 6D- E).
  • T cell subsets were further evaluated in the spleen, with CD3+, CD4+ single positive, CD8+ single positive, CD4+CD8+ double positive, CD4+CD25+FOXP3+ Tregs, memory CD4+CD45RA-, and naive CD4+CD45RA+ T cells all present in both FOXP3FLcoW edited (tNGFR+ and tNFGR- fraction) and control mice (Fig. 6D- E). Some differences in the frequencies of cell subsets were observed between conditions, such as higher proportion of CD3+ cells and a lower proportion of CD25+FOXP3+ cells in the tNGFR+ fraction. However no overt changes to immune reconstitution were observed and each hematopoietic cell lineage was represented among the different experimental conditions (Fig. 6E).
  • Tregs and Teff cells derived from in vivo differentiation of edited HSPCs we sorted CD3+CD4+ Tcells from hu-mouse spleens and separated them into CD25high (Treg) and CD25low (Teff) fractions. The Treg and Teff fractions from the edited conditions were further sorted into tNGFR+ and tNGFR- fractions.
  • Persistent expression of FOXP3 in Tregs preserved their phenotype and ability to suppress T cell function.
  • the level of FOXP3 expression in edited Teff cells closely mirrored that of WT cells, while edited Tregs displayed partial FOXP3 protein expression. This cell type distinct result could be attributed to the fact that Tregs physiologically express a much higher level of FOXP3 than activated Teff cells.
  • the level of FOXP3 expression in edited Tregs led to average suppressive function less than that of WT Tregs from normal donors, the suppressive rates of edited cells were still within the lower range of non- edited normal donor function.
  • the range in suppressive function among healthy donor Tregs highlights the variability of in vitro regulatory function among individuals. Importantly, restoration of a similar level of FOXP3 expression in IPEX patient Tregs was sufficient to reestablish suppressive activity in co-culture with Teff cell responders.
  • transgene promoter and enhancer elements may raise concerns of inadvertently activating proto-oncogenes or genes that would be detrimental to hematopoiesis. These concerns underscore the benefits of site-specific gene editing as a more precise method for therapeutic gene delivery.
  • the CRISPR system enabled efficient HDR-mediated editing of FOXP3 in HSPCs, the cell type used for autologous HSPC transplantation and long term reconstitution of the immune system.
  • the overall targeted integration frequency was 29 ⁇ 8% when using tNGFR marker to identify targeted cells.
  • targeting frequency increased to 50% or greater when the HDR donor was shortened by removal of the tNGFR marker gene. While the tNGFR marker facilitates enrichment of edited cells for functional testing, it may not always be necessary in clinical settings. The marker gene could potentially be removed to improve editing rates if it becomes apparent that selection and tracking of tNGFR expressing cells is not essential.
  • HSPCs can be used for autologous transplant, or CRISPR system could be applied to adoptive cell therapy using differentiated T cells or T cell precursor cells. Additionally, the efficient CRISPR-based editing of T cells facilitated functional testing of edited Tregs and Teff cells.
  • the T cell functional assays performed in this study used the codon optimized divergent FOXP3 cDNA sequence followed by a WPRE element, which was expected to provide optimal protein expression. Different codon diverged sequences can be used in Tregs, and modifications to the CG content of the cDNA could be examined. Expression from the endogenous FOXP3 gene and translation of FOXP3 protein may be enhanced by endogenous intron-exon splice sites and 3’UTR elements. Modifications to the construct aimed at improving expression can include incorporation of short exogenous intronic sequences or 3’UTRs from genes highly expressed in Tregs.
  • FOXP3F L and FOXPS ⁇ 2 both isoforms may be required to reach wild-type levels of FOXP3 expression in Tregs.
  • CRISPR-mediated knock-in uses endogenous FOXP3 promoters and enhancers and allows each isoform to be expressed individually under physiological conditions. Although expression of each isoform was only -50-60% of wild-type levels, each isoform alone at that level was able to support suppressor function within the lower range of healthy donor cells.
  • the ability to deliver individual isoforms into the endogenous locus allows the FOXP3 CRISPR system to be used as a tool to investigate the FOXP3F L and FOXP3 dE2 isoforms individually.
  • co-delivery of both FO P3 FL and FOXP3 dE2 cDNAs and subsequent simultaneous expression of both isoforms in may find use.
  • the FOXP3 CRISPR system was similarly used as a tool to study the effects of complete FOXP3 knockout (FOXPS ⁇ 0 ). We observed that FOXP3 loss in Tregs ablated suppressive function.
  • the tNGFR marker was used to isolate a pure population of FOXP3 KO cells for functional analysis. The ability to purify FOXP3 null cells makes this approach superior to an incomplete knockdown or heterogeneous indel-mediated knockout of FOXP3 as previously used to investigate the effects of FOXP3 loss.
  • FOXP3 CRISPR system allows for the creation of IPEX-like cell models in more readily available healthy donor cells.
  • the CRISPR system can be used to knock-in FOXP3 cDNAs harboring patient-specific mutations, that provide insight into the molecular mechanisms underlying the heterogeneity of clinical presentation in IPEX syndrome.
  • FOXP3 loss in both FOXP 0 and IPEX Tregs abrogated suppressive function
  • FOXP3 cDNA knock-in Tregs displayed in vitro function and maintained characteristic Treg phenotypic markers.
  • Transplanted IPEX patients with low overall chimerism of donor cells can still undergo tremendous disease regression, especially due to the fact that the Treg compartment shows a selective advantage toward donor cells.
  • carrier mothers display a selective advantage in the Treg compartment such that the mutated FOXP3 allele is predominantly located in the inactivated X chromosome, while the wild-type allele of FOXP3 is expressed from the active X chromosome in the Treg compartment.
  • hu-mouse-derived FOXP3 edited Teff cells were found to be functional in vitro and proliferate at comparable rates to WT Teff cells. These functional assays demonstrate that FOXP3 edited HSPCs retain the capacity to give rise to functional Tregs and Teff cells in vivo.
  • the HSPCs were cultured at 37°C with 5% CO 2 and low oxygen (5% O 2 ) in StemSpan SFEM II medium (StemCell Technologies) supplemented with 100 ng/ml SCF (PeproTech), 100 ng/ml IL-6 (PeproTech), 100 ng/ml TPO (PeproTech), 100 ng/ml Flt3L (PeproTech), 750 nM StemRegeninl (StemCell Technologies), and 35nM UM171 (StemCell Technologies). Additional healthy donor Treg and Teff cells were obtained from the Stanford Blood Center, and peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque density gradient separation.
  • PBMCs peripheral blood mononuclear cells
  • Tregs and Teff cells were separated by magnetic bead isolation using the CD4+CD25+ Regulatory T Cell Isolation Kit (Miltenyi Biotec) according to the manufacturer’s protocol. Both cell fractions were activated with 10 ug/ml immobilized plate-bound anti-CD3 (OKT3 mAb, Miltenyi) with soluble 1 ug/ml anti- CD28 mAb (NA/LE, BD) for 2 to 3 days prior to editing and then switched to non-activation conditions.
  • Tregs were cultured in X-VIVO 15 (Lonza) with 5% human serum from male AB plasma (Sigma- Aldrich), 300 U/ml IL-2 (PeproTech), and 100 nmol rapamycin (StemCell Technologies, only added for certain experiments). Teff cells were cultured in X-VIVO 15, 5% human serum, and 50 U/ml IL-2. The Tregs and Teff cells were cultured at 37°C with 5% CO2 and ambient oxygen levels. Treg-like MT-2 cells were cultured in X-VIVO 15 with 5% human serum and 1% penicillin/streptomycin.
  • K562 cells were cultured in RPMI medium (Thermo Fisher) with 10% FBS (Fisher Scientific) at 37°C with 5% CO2 and ambient oxygen levels. For all cells, fresh medium was added every 2 to 3 days.
  • CRISPR chimeric sgRNA were designed using the Desktop Genetics web based tool (MIT) and cloned into expression vectors using the px330 plasmid backbone (Addgene). The sgRNAs were placed under the human U6 promoter in the px330 plasmid, which also contained an expression cassette for human codon-optimized SpCas9.
  • sgRNAs were cloned into the px335 plasmid (Addgene) containing the Cas9 nickase expression cassette, and were codelivered as paired plasmids.
  • px335 plasmid containing the Cas9 nickase expression cassette
  • paired plasmids 2ug of sgRNA/Cas9 plasmid DNA was nucleofected into 1 million K562 cells using the Lonza Nucleofector 2b (program T-016).
  • 100 uL of nucleofection solution was used (100 mM KH2PO4, 15 mM NaHC03, 12 mM MgCl2- 6H2O, 8 mM ATP, 2 mM glucose (pH 7.4)).
  • the cells were cultured for 2-4 days and genomic DNA was extracted using QuickExtract DNA Extraction Solution (Epicentre) according to manufacturer’s recommendations.
  • the site of DNA cleavage was PCR amplified using Herculase II fusion polymerase (Aligent Technologies) and primers flanking the region 5’-CTAGAGCTGGGGTGCAACTATGG-3’ and 5’- GACTACAATACGGCCTCCTCCTCTC-3’.
  • the PCR amplicons were gel purified (Qiagen) and sequenced by Sanger sequencing using the forward primer 5’- CTAGAGCTGGGGTGCAACTATGG-3’.
  • the resulting sequences were used to calculate indel frequencies using the TIDE analysis web based software (https://tide.nki.nl/).
  • Table 1 A list of all sgRNA and primer sequences is provided in Table 1.
  • FOXP3 homology donor design The FOXP3 cDNA sequence was modified, or diverged, to incorporate synonymous mutations at the nucleotide level according to the redundant codon usage system to prevent premature recombination while still encoding for the wild-type FOXP3 protein.
  • a constitutive Phosphoglycerate Kinase (PGK) promoter was positioned upstream of the tNGFR gene such that the marker would be expressed in all edited cells independent of FOXP3 expression.
  • a strong Bovine Growth Hormone (BGH) polyadenylation signal (pA) was positioned after the FOXP3 cDNA, and another pA was included after the tNGFR marker gene to allow independent expression of the FOXP3 cDNA and tNGFR, and to ensure inactivation of the remaining endogenous FOXP3 locus.
  • the homology arms were centered on the cut site of the sgRNA 2.
  • the 3’arm (right arm) started at the cut site and reached approximately 650bp downstream of the cut site, whereas the 5’ arm (left arm) included a region approximately 600 bp upstream of the cut site.
  • the FOXP3F LcoW construct contained a shorter synthetic pA site in place of the BGH pA and shorter arms of homology to accommodate the addition of a WPRE element while maintaining an overall similar donor length.
  • pAAV-FOXP3 plasmids were co- transfected with rAAV6 helper plasmid DNA into the 293FT Cell Line (Life Technologies). After 72hr, rAAV6-FOXP3 viral particles were extracted using the AAVpro kit (Clontech, Takara) according to manufacturer’s instructions. The viral stocks were titered using qPCR with primers and probe annealing to the ITRs.
  • the rAAV genomic DNA was isolated using QIAamp MinElute Virus Spin Kit (Qiagen), qPCR was performed on the Roche LightCycler 480, and viral titer (vector genomes per uL) was calculated using a standard curve generated from a circular pAAV-MCS-donor plasmid of known concentration.
  • Gene editing by nudeofection and rAAV transduction was performed using synthetic sgRNA 2 (5’- AGGACCCGATGCCCAACCCC-3’) complexed to SpCas9 protein (IDT) as an RNP system.
  • the sgRNA was synthesized as a 100-mer RNA molecule with 2'-0-methyl 3'phosphorothioate (MS) chemical modifications at the three terminal nucleotides on the 5' and 3' ends (SEQ ID NO:2) (5’- 2OMe(A(ps)G(ps)G(ps))ACC CGA UGC CCA ACC CCG UUU UAG AGC UAG AAA UAG CAA GUU AAA AUA AGG CUA GUC CGU UAU CAA CUU GAA AAA GUG GCA CCG AGU CGG UGC UUU 2’0Me(U(ps) U(ps)U)-3’, ps indicates phosphorothioate, 2OMe indicates 2'-0-methyl).
  • MS 3'phosphorothioate
  • the sgRNAs were purified by reversed phase high-performance liquid chromatography (HPLC) and quantified by mass spectrometry.
  • HPLC reversed phase high-performance liquid chromatography
  • the sgRNAs were purchased from TriLink Biotechnologies, and in later experiments, from Synthego (not HPLC purified), and editing rates triggered by sgRNAs from the two respective companies were comparable when tested in parallel.
  • the sgRNA was complexed with Cas9 for 10min at 25°C at an approximate Cas9:sgRNA molar ratio of 1 :2.5, using 8ug of sgRNA and 15ug of Cas9 per 100uL nudeofection solution containing 2.5x10 5 to 1x10 6 cells.
  • HiFi Cas9 After switching to high fidelity (HiFi) Cas9 (IDT) that showed slightly lower efficiency in parallel experiments, the amount of HiFi Cas9 was increased and the molar ratio was adjusted to 1 :1 .8, using 8ug of sgRNA with 22ug of HiFi Cas9 per 100uL nudeofection solution.
  • the sgRNA/Cas9 complexes were nucleofected into Tregs and Teff cells after 2-3 days of activation using the P3 Primary Cell Nudeofection Kit (Lonza) and the Lonza Nucleofector 4D (program E-0115). On the day of nudeofection, additional antibiotic (Pen/Strep) was removed from the medium, and rapamycin was removed from the Treg medium.
  • HSPC editing the cells were nucleofected using the P3 Primary Cell Nudeofection Kit (Lonza) and the Lonza Nucleofector 4D (program DZ- 100).
  • genomic DNA was extracted using QuickExtract DNA Extraction Solution (Epicentre) and TIDE analysis was performed as described above.
  • rAAV6-FOXP3 donor transduction was performed following nudeofection at an MOI of 1x10 5 to 1 x10 6 viral genomes per cell. After 24hr of transduction, the medium was changed to remove excess viral particles.
  • Herculase II fusion polymerase (Aligent Technologies) was used for all PCR amplification steps. The resulting PCR products were resolved by agarose gel electrophoresis. For absolute quantification of genomic integration events at the DNA level, an in-out PCR strategy quantified using the Digital Droplet PCR (ddPCR, BioRad) system was used.
  • ddPCR Digital Droplet PCR
  • tNGFR+ edited cells Enrichment of tNGFR+ edited cells. Edited tNFGR+ cells were enriched by fluorescence- activated cell sorting (FACS) 2-4 days post-editing on a FACS Aria II SORP (BD Biosciences). Cells were stained with anti- NGFR/CD271 mAb (Biolegend, clone ME20.4, PE-Cy7- conjugated or APC-conjugated). When edited cells were present at low cell numbers, magnetic bead cell isolation was used to increase yield and avoid cell loss associated with FACS sorting. Positive selection of tNGFR+ cells was performed using CD271 (tNGFR) Microbead Kit (Miltenyi Biotech) according to the manufacturer’s instructions.
  • FACS fluorescence- activated cell sorting
  • the px330-FOXP3-sgRNA2-Cas9 plasmid was electroporated into U20S cells along with a double-stranded oligodeoxynucleotide (dsODN, 5'- GTTTAATTGAGTTGTCATATGT- TAATAACGGTAT-3').
  • dsODN double-stranded oligodeoxynucleotide
  • the T7 and RFLP assays were performed to confirm editing and tag integration (using TIDE primers 5'-
  • NGS sequencing reads that were identified at similar rates in edited cells and mock treated samples were eliminated from the analysis. High background in the mock treated samples was attributed to the proximity of the sequencing primer to the polynucleotide sequence 5’-CCCC- 3’ in the sgRNA target site, as polyN sequences commonly cause errors in NGS and can lead to false positive indel identification.
  • FOXP3 mRNA expression was detected by RT-PCR in FOXP3 ⁇ edited CD4+ T cells and controls after 3 days of re-activation with Human T-Activator anti-CD3/28 Dynabeads (Life Technologies, 1 :25 bead:cell ratio).
  • RNA was extracted with TriReagent (Sigma-Aldrich) and polyA+ mRNA was reverse transcribed into cDNA using Superscript III First-Strand Synthesis System (ThermoFisher).
  • PCR amplification of FOXP3 cDNA was performed using Herculase II fusion polymerase (Aligent Technologies) and primers listed in Table 1.
  • FOXP3 For assessing FOXP3 expression by flow cytometry, cells were fixed and permeabilized using FOXP3 staining solutions (eBioscience) and stained with anti-FOXP3 mAb (clone 259D/C7) conjugated to either AF647 (BD Biosciences) or AF488 (Biolegend) following manufacturer’s instructions. Fluorescence was detected on a FACS Aria II SORP (BD Biosciences), analyzed using FlowJo software v4 10.5.0, and median florescent intensity (MFI) was recorded.
  • FOXP3 staining solutions eBioscience
  • anti-FOXP3 mAb clone 259D/C7 conjugated to either AF647 (BD Biosciences) or AF488 (Biolegend) following manufacturer’s instructions. Fluorescence was detected on a FACS Aria II SORP (BD Biosciences), analyzed using FlowJo software v4 10.5.0, and median florescent intensity (MFI) was recorded.
  • Treg phenotyping and suppression assay For Treg phenotyping, cells were stained for flow cytometry using the following antibodies: CD25-BV605 (clone 2A3, BD Biosciences), CTLA-4-PerCPCy5.5 (L3D10, BioLegend), FOXP3- AF647 (259D/C7, BD), HELIOS-PE (22F6, Biolegend), NGFR-BV421 (cME20.4, BioLegend), PD-1-FITC (MIH4, BD), and TIGIT- PE-Cy7 (MBSA43, eBioscience).
  • Intracellular staining for FOXP3, CTLA-4, and HELIOS was performed after fixing and permeabilizing with FOXP3 staining solutions (eBioscience). Expression was detected on a FACS Aria II SORP (BD Biosciences) and geometric mean intensity was analyzed using FlowJo software v4 10.5.0. The function of gene edited Tregs was tested by the suppression assay using allogenic CD4+ T cell responders that were labeled with CFSE proliferation dye (CellTrace CFSE Cell Proliferation Kit, Life Technologies).
  • Responders were plated at a concentration of 2 x 10 4 cells/well and co- cultured with Tregs at a 1 :1 or 1 :0.5 ratio of responders:suppressors.
  • the cells were activated with a 1 :25 ratio of beads:cells using Human T-Activator anti-CD3/28 Dynabeads (Life Technologies).
  • responders were co-cultured with an equal number of unstained Teff cells.
  • the cells were cultured in 96-well round well plates and analyzed for CFSE staining on days 3-5 using a FACS Aria II SORP (BD Biosciences).
  • Non-activated responders were used for gating and the percent of proliferated cells was analyzed using FlowJo software v4 10.5.0.
  • % suppression ((%proliferated R * ) - (%proliferated R * +Treg)) / (%proliferated R * ) x 100), where R * represents stimulated CFSE-stained responder Teff.
  • Treg purity was performed by flow cytometry analysis using the following antibodies: CD4-APC-Cy7 (RPA-T4, Biolegend), CD25- PE (4E3, Miltenyi), CD127- PerCP-Cy5-5 (A019D5, Biolegend), and FOXP3-AF647 (clone 259D/C7, BD).
  • the frequency of demethylated TSDR Tregs was quantified by epigenetic bisulfite qPCR in collaboration with Epimune/Epiontis GmbH (Berlin, Germany) as previously described.
  • Teff cytokine quantification and proliferation assay Teff cytokine quantification and proliferation assay. Teff cytokine production was quantified using ELISA for IL-2 (BD Biosciences), IFN-Y (BD Biosciences), and IL-17 (R&D Systems). Teff cells were activated using Human T-Activator anti-CD3/28 Dynabeads (Life Technologies) at a 1 :25 ratio of beads:cells in 96-well round well plates at 2 x 10 s cells per 200 uL. Supernatants were collected at 24hr (IL-2) and 48hr (IFN-D and IL-17) post-activation.
  • IL-2 Human T-Activator anti-CD3/28 Dynabeads
  • Teff cells were stained using the CellTrace CFSE Cell Proliferation Kit (Life Technologies) and cultured at 5 x 10 4 cells/well in 96-well round well plates. The stained cells were activated with a 1 :25 ratio of anti-CD3/28 Dynabeads and analyzed for CFSE staining on days 2-4 post-activation on a FACS Aria II SORP (BD Biosciences). The percentage of proliferated cells was determined using FlowJo software v4 10.5.0 and gated using non-activated responders as a reference.
  • CFU assay to assess in vitro HSPC differentiation.
  • Gene edited cord blood-derived HSPCs were FACS sorted 2-4 days post-editing and differentiated in vitro using the colony forming unit (CFU) assay.
  • CFU colony forming unit
  • 500 cells were plated in 1.1 ml. of semi-solid methylcellulose medium (Methocult H4434, StemCell Technologies) and performed in duplicate or triplicate.
  • the cells suspended in methocult were incubated at 37°C with 5% C0 2 and ambient oxygen levels, and the resulting progenitor colonies were counted and scored after 14 days (BFU-E (primitive erythroid progenitors), CFU-E (mature erythroid progenitors), CFU-GM (granulocyte and macrophage progenitors), and CFU-GEMM (granulocyte, erythrocyte, macrophage, megakaryocyte)).
  • BFU-E primary erythroid progenitors
  • CFU-E mature erythroid progenitors
  • CFU-GM granulocyte and macrophage progenitors
  • CFU-GEMM granulocyte, erythrocyte, macrophage, megakaryocyte
  • HSPCs were phenotyped by flow cytometry to ensure purity using the antibodies, CD34-PE-Cy7 (4H11 , eBiosciences), CD38-Percp-Cy5.5 (HIT2, BioLegend), CD45RA-FITC (H1100, BD Biosciences), CD90-APC- Cy7 (5E10, BioLegend), CD49f-PE (GoH3, BioLegend), and combined lineage markers (Lin) on APC as follows: CD45-APC (30-F11 , BioLegend), CD19-APC (HIB19, BioLegend), CD14- APC (HCD14, BioLegend), CD235a-APC (HIR2, BioLegend), CD20-APC (2H7, BioLegend), CD16-APC (3G8, BioLegend), CD2-APC (RPA- 2.10, BioLegend), CD3-APC (SK7, Biolegend), CD4-APC (SK3, Biolegend), CD
  • mice were checked for peripheral engraftment of human CD45+ cells via biweekly retroorbital bleed. The mice and were sacrificed between 11-14 weeks, and blood, spleen, bone marrow, and thymus were harvested.
  • red blood cells were lysed following a 5 min incubation on ice with 1x RBC lysis buffer (eBiosciences) and were resuspended in staining buffer (PBS, 0.25% BSA, 1 mM EDTA).
  • Cells purified from tissues were stained using the following antibodies: hCD45 BV510 (HI30, BD Biosciences), mCD45- APC (30-F11 , BioLegend), CD3-Percp Cy5.5 (OKT3, BioLegend), CD56- PE (5.1 H11 , Biolegend), CD13-APC-Cy7 (WM15, BioLegend), and CD19-FITC (HIB19, BD Biosciences).
  • An additional antibody panel for T cell subsets included: hCD45 BV510 (HI30, BD Biosciences), mCD45 PE (30-F11 , BioLegend), CD4 APC-Cy7 (RPA-T4, BioLegend), CD8 BV650 (SK1 , BioLegend), CD25 BV605 (2A3, BD Biosciences), CD45RA FITC (HI100, BD Biosciences), and FOXP3 AF647 (259D, BioLegend).
  • Cell were either analyzed by flow cytometric analysis (CytoFLEX BD) or sorted (BD, FACSAria). Sorted CD25 high and CD25 low populations were analyzed by suppression and proliferation assays, respectively, as described above.
  • MPNPRPGKPS APSLALGPSP GASPSWRAAP KASDLLGARG PGGTFQGRDL RGGAHASSSS LNPMPPSQLQ LPTLPLVMVA PSGARLGPLP HLQALLQDRP HFMHQLSTVD AHARTPVLQV HPLESPAMIS LTPPTTATGV FSLKARPGLP PGINVASLEW VSREPALLCT FPNPSAPRKD STLSAVPQSS YPLLANGVCK WPGCEKVFEE PEDFLKHCQA DHLLDEKGRA QCLLQREMVQ SLEQQLVLEK EKLSAMQAHL AGKMALTKAS SVASSDKGSC CIVAAGSQGP VVPAWSGPRE APDSLFAVRR HLWGSHGNST FPEFLHNMDY FKFHNMRPPF TYATLIRWAI LEAPEKQRTL NEIYHWFTRM FAFFRNHPAT WKNAIRHNLS LHKCFVRVES EKGAVWTVDE LEFRKKRSQ
  • NCBI Reference Sequence NP 001107849.1 (SEQ ID N0:2
  • MPNPRPGKPS APSLALGPSP GASPSWRAAP KASDLLGARG PGGTFQGRDL RGGAHASSSS LNPMPPSQLQ LSTVDAHART PVLQVHPLES PAMISLTPPT TATGVFSLKA RPGLPPGINV ASLEWVSREP ALLCTFPNPS APRKDSTLSA VPQSSYPLLA NGVCKWPGCE KVFEEPEDFL KHCQADHLLD EKGRAQCLLQ REMVQSLEQQ LVLEKEKLSA MQAHLAGKMA LTKASSVASS DKGSCCIVAA GSQGPVVPAW SGPREAPDSL FAVRRHLWGS HGNSTFPEFL HNMDYFKFHN MRPPFTYATL IRWAILEAPE KQRTLNEIYH WFTRMFAFFR NHPATWKNAI RHNLSLHKCF VRVESEKGAV WTVDELEFRK KRSQRPSRCS NPTPGP
  • F0XP3 Full Length Codon Diverged Gene Editing Construct (SEQ ID NO:3). nt. 1-8 Not I site; nt. 9-625 5’ homology arm; nt. 616-1912 FOXP3 coding sequence (encoding SEQ ID NO:1); nt. 1913-2139 BGH poly-A signal; nt. 2140-2660 PGK promoter; nt. 2661-3503 tNGFR marker sequence; nt. 3504-3730 BGH poly-A signal; nt. 3731 -4386 3’ homology arm; nt. 4387- 4394 Not I site.
  • FOXP3 D2 Codon Diverged Gene Editing Construct (SEQ ID NO:4).
  • the features are as in SEQ ID NO:3, with the exception that the shorter isoform FOXP3 coding sequence is nt. 616- 1807, encoding SEQ ID NO:2
  • FOXP3 in-out PCR primers and probes for quantitative ddPCR analysis SEQ ID NO:27 FOXP3 control ddPCR RP 5’-CCCGGGGGAGTATAGAAGG-3’

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Abstract

L'invention concerne des compositions et des procédés se rapportant à des cellules hématopoïétiques modifiées par le gène FOXP3, comprenant des cellules souches et progénitrices hématopoïétiques, des cellules progénitrices lymphoïdes et des lymphocytes T CD4+. Les cellules modifiées par un gène sont utiles dans la thérapie cellulaire pour restaurer des fonctions immunitaires normales et favoriser la tolérance immunitaire. En particulier, les lymphocytes T CD4edFOXP3, qui peuvent être différenciés à partir de cellules progénitrices hématopoïétiques modifiées par le gène FOXP3, peuvent exprimer physiologiquement FOXP3 fonctionnel et exercer des réponses immunitaires normales en tant que lymphocytes T effecteurs ou ont des caractéristiques immunosuppressives comme lymphocytes T régulateurs d'origine naturelle.
PCT/US2021/018057 2020-02-13 2021-02-13 Lymphocytes t modifiés par un gène foxp3 à base de crispr et précurseurs de cellules souches hématopoïétiques permettant de traiter des patients atteints de syndrome ipex WO2021163642A2 (fr)

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CA3168089A CA3168089A1 (fr) 2020-02-13 2021-02-13 Lymphocytes t modifies par un gene foxp3 a base de crispr et precurseurs de cellules souches hematopoietiques permettant de traiter des patients atteints de syndrome ipex
US17/760,264 US20230081343A1 (en) 2020-02-13 2021-02-13 Crispr-based foxp3 gene engineered t cells and hematopoietic stem cell precursors to treat ipex syndrome patients

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CN113403208A (zh) * 2021-06-15 2021-09-17 江西科技师范大学 高效鉴定米曲霉CRISPR/Cas9突变体的方法
WO2023122099A3 (fr) * 2021-12-21 2023-08-03 The Board Of Trustees Of The Leland Stanford Junior University Édition de gènes basée sur crispr pour préserver l'épissage et l'expression d'isoformes de foxp3 1 et 2
WO2023205657A3 (fr) * 2022-04-18 2023-11-23 City Of Hope Compositions pour restaurer la fonction du gène mecp2 et leurs méthodes d'utilisation

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CA2339409A1 (fr) * 1998-08-11 2000-02-24 Darwin Discovery Ltd. Identification du gene responsable du phenotype de la souris "scurfy" et de son orthologue humain
US8501464B2 (en) * 2003-04-24 2013-08-06 Ospedale San Raffaele S.R.L. Lentiviral vectors carrying synthetic bi-directional promoters and uses thereof
CA3091491A1 (fr) * 2018-04-27 2019-10-31 Seattle Children's Hospital (dba Seattle Children's Research Institute) Expression de foxp3 humain dans des lymphocytes t a edition genique
AU2019261438A1 (en) * 2018-04-27 2020-09-10 Seattle Children's Hospital (dba Seattle Children's Research Institute) Expression of FOXP3 in edited CD34+ cells

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113403208A (zh) * 2021-06-15 2021-09-17 江西科技师范大学 高效鉴定米曲霉CRISPR/Cas9突变体的方法
WO2023122099A3 (fr) * 2021-12-21 2023-08-03 The Board Of Trustees Of The Leland Stanford Junior University Édition de gènes basée sur crispr pour préserver l'épissage et l'expression d'isoformes de foxp3 1 et 2
WO2023205657A3 (fr) * 2022-04-18 2023-11-23 City Of Hope Compositions pour restaurer la fonction du gène mecp2 et leurs méthodes d'utilisation

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